ACTIVE AND ACID-ACTIVATABLE TGF-BETA IN HUMAN SERA, PLATELETS AND PLASMA

Assays which measure active and latent forms of transforming growth factor beta (TGF-beta) separately in human serum and plasma are required to investigate the biological role of TGF-beta in a variety of human diseases. We have developed an enzyme-linked immunosorbent assay (ELISA) using two polyclonal antibodies against TGF-beta which rapidly determines the amount of active plus acid-activatable, latent TGF-beta forms ((a+1)TGF-beta) present in human serum and plasma in the range 4 pmol/l to 2000 pmol/l. To measure active TGF-beta alone, we have developed a second ELISA using the extracellular domain of the TGF-beta type II receptor as the capture reagent which detects active TGF-beta in serum and plasma samples in the range 20 pmol/l to 4000 pmol/l. Both assays detect TGF-beta 1 and TGF-beta 3 with similar sensitivity, are > 10-fold less sensitive to TGF-beta 2 and are not affected by a range of other peptide growth factors. The mean (a+1)TGF-beta present in human serum was 330 pmol/l but the range was very large(<4 pmol/l to 1400 pmol/l). The mean active TGF-beta present was 230 pmol/l (range <20 pmol/l to 1400 pmol/l) and the proportion of the (a+1)TGF-P present which was active [a/(a+1)] varied from < 10% to 100%. The concentration of(a+1)TGF-beta and the proportion of TGF-beta which was active were very similar in the serum and platelet-poor plasma prepared from the same whole blood sample. The clot formed during serum preparation retained all of the TGF-beta which was detected by the (a+1)TGF-beta ELISA in the corresponding platelet releasate, although the PDGF in platelets was released into the serum. In contrast, platelet-poor plasma contained no detectable PDGF demonstrating that the (a+1)TGF-beta assayed in the plasma was not due to platelet degranulation after bleeding. Serum active TGF-beta and (a+1)TGF-beta concentrations therefore provide a reliable estimate of these forms of TGF-beta present in plasma.